Immunogenicity and efficacy of fourth BNT162b2 and mRNA1273 COVID-19 vaccine doses; three months follow-up

Booster doses for the ongoing COVID-19 pandemic are under consideration in many countries. We report a three-month follow-up of 700 participants in a fourth vaccine dose study, comparing BNT162b2 and mRNA1273, administered four months after a third BNT162b2 dose. The primary outcomes are the levels of IgG, neutralizing antibodies, and microneutralization and the secondary outcomes are the levels of IgA and T cell activation, and clinical outcomes of SARS-CoV-2 infection and substantial symptomatic disease. Waning of the immune response is evident during follow-up, with an 11% (β = 0.89, 95% CI, 0.88–0.9) and 21% (β = 0.79, 95% CI, 0.76–0.82) multiplicative decay per week of IgG and neutralizing antibodies, respectively, in the mRNA1273 group, and of 14% (β = 0.86, 95% CI, 0.86–0.87) and 26% (β = 0.74, 95% CI, 0.72–0.76), respectively, in the BNT162b2 group. Direct neutralization of Omicron variants is low relative to ancestral strains. Cumulatively over the study period, both vaccines show little efficacy against infection but were highly efficacious against substantial symptomatic disease [89% [(IRR 0.11, 95% CI, 0.02–0.37) and 71% (IRR 0.29, 95% CI, 0.13–0.57) for mRNA1273 and BNT162b2, respectively]. These results are informative for further boosting policy-making. Trial registration numbers (clinicaltrials.gov): NCT05231005 and NCT05230953.

3 plates for 60 minutes at 33ºC. Virus-serum mixtures were added to the Vero E-6 cells and incubated for five days at 33ºC after which Gentian violet staining (1%) was used to stain and fix the cell culture layer. Neutralizing dilution of each serum sample was determined by identifying the well with the highest serum dilution without observable cytopathic effect. A dilution equal to 1:10 or above was considered neutralizing.

IgA assay
IgA antibodies against the S1 domain of the spike protein of SARS-CoV-2 (expressed recombinantly in the human cell line HEK 293) were detected by a semiquantitative enzyme-linked immunoassay (ELISA) (anti-SARS-CoV-2 ELISA IgA, Euroimmun, Lübeck, Germany). Samples were tested according to the manufacturer's instructions. For the ELISA, a 96 well microtiter Polysorb plate (Nunc, Thermo, Denmark) was coated overnight at 4°C with 50µl per well of 2µg/ml. After blocking with 5% skimmed milk at 25°C for 60 minutes, positive and negative control and human serum samples without replications and calibrator (sample at the cut-off O.D value) in triplicate (all diluted 1:100 with 3% skimmed milk), were added to antigen coated wells. The plate was incubated at 25°C for 120 minutes, washed and a HRP-conjugated isotype specific antibody (anti-human IgA horseradish peroxidase (HRP) conjugate (Abcam, MA, USA, product number: ab7383) (diluted 1:2000)) was added to each well for 60 min. After washing, incubation of TMB Substrate Solution (Abcam) for 5 min and the addition of stop solution (2N HCl), the OD of each well was measured at 450nm using a micro-plate reader (Sunrise, Tecan).
ELISA index value was defined as the ratio between sample and cut-off optical densities (OD). ELISA index value below 0.9 was considered negative, between 0.9 and 1.1, borderline and equal or above 1.1, positive.
PCR tests were performed according to the manufacturer's instructions using Allplex™ 2019-nCoV (Seegene, S. Korea) platform. Rapid antigen tests were performed according to manufacturer's instructions using STANDARD Q COVID-19 Ag (SD BIOSENSOR, S. Korea).
Cases were defined as positive also when an uncharacteristic increase in IgG levels was observed. We previously reported a substantial increase in IgG levels following vaccination or infection which was maintained for 30 days, after which a slow consistent decline was observed. Therefore, an uncharacteristic increase in IgG levels of >500 BAU or >1000 BAU in HCW with previous IgG results of <700 or >700, respectively was considered as a sero-response due to SARS-CoV-2 infection (3)(4)(5).  11 11 (1-142) Immunoglobulin G (IgG) and neutralizing antibody titers are presented in binding antibody units (BAU), Immunoglobulin A (IgA) is presented as sample-to-cutoff (s/co) ratio, T cells are presented as activated T cells per 10 6 peripheral blood mononuclear cells (PBMC).

Supplementary Table S6-Variable definitions
IgG-anti-RBD immunoglobulin G, IgA-anti-RBD immunoglobulin A, GMT-geometric mean titers, SARS-CoV-2-severe acute respiratory syndrome coronavirus 2, qRT-PCR-quantitative real-time polymerase chain reaction, Ag-RDT-antigen rapid diagnostic test, BMI-body mass index, HIV-human immunodeficiency virus. *Uncharacteristic increase in IgG levels was defined as >500 BAU or >1000 BAU in HCW with previous IgG results of <700 or >700, respectively. We compared this to 3-dose vaccinated, and also showed a significantly reduced neutralization of early 3-dose vaccinated as compared to the efficiency against Wu-1 or the delta VOC. Furthermore, data from Germany showed that neutralization of Omicron decreases within 3 months following the 3 rd dose.
These data raise the question of when and will we need a 4 th dose to cope with the emergence of Omicron. However, if we have reached the maximal effect of the current vaccine against Omicron, with a third dose, will a 4 th dose have any added value? In this respect, several studies have demonstrated improved immunogenicity of 1 vaccine dose to SARS-CoV-2 recovered patients, yet a 2 nd dose to these patients had very little added value. It is thus unclear whether a 4 th dose of the current vaccine, which is not directed specifically at the Omicron VOC, can further improve our immunity and protection from this strain, and will it have any added value.
The Sheba HCW COVID Cohort was established on April 2020, when HCW (including employees, students, volunteers and retired personnel), were recruited to a large serology longitudinal follow up study. Participation in the study is confidential and is not disclosed to the worker's direct supervisor. Since then, nearly 7000 HCW who were vaccinated by BNT162b2 are being followed monthly with blood sampling for immune responses, including mostly IgG, but also Neutralizing ab, avidity, microneutralization, IgA and cellular activation. Through this cohort we found the correlation between neutralizing ab levels and SARS-CoV-2 infections (Bergwerk et al., 2021) , the waning of immunity (Levin et al., 2021)and more. In our study, the median neutralizing ab titer of cases was 190, while it was significantly higher (530) among controls. Furthermore, (Immune Correlates Analysis of the MRNA-1273 COVID-19 Vaccine Efficacy Clinical Trial, n.d.) report that titer of 1000 or more are correlated with 96% vaccine efficacy and a titer of 100 is correlated with 91%, but this is for the era before Omicron. We and others have shown a decreased efficacy for Omicron of 8fold to 24-fold compared to WT.
Here, we will study the potential immunogenicity of a 4 th dose, together with assessing safety and efficacy in preventing infections.

Study Design
This is an open-labled, controlled intervention trial. Two intervention arms are planned and participants will be designated to each arm on a time dependent manner. Eligible participants are those enrolled to the Sheba COVID-19 Cohort study (IRB 8008-20), who have been followed for serology testing, have an available test from the recent 60 days with a result of IgG of 700BAU or below (i.e., under 40 percentile of the full cohort on Dec 2021) and have received the third BNT162b2 dose at least 4 months earlier (see below full inclusion/exclusion criteria). Initially volunteers will be designated to the BNT162b2 arm, and once approval of the second arm will be received, volunteers will be designated to the second -mRNA1273 arm. Age matched controls, who are eligible to be enrolled to the intervention arm will be selected for the efficacy analysis. Figure 1: Flow Chart of eligibility and study population selection Background comorbidity X Adverse events X X

Primary outcomes:
Serology tests as detailed below, including IgG, pseudoneutralization, microneutralization and avidity. These outcomes were compared between pre-and post-4 th dose as well as with those outcomes in the second intervention arm and in the control group.
Adverse event reporting of vaccinated individuals by an electronic survey that will be on day 5 and will be repeatedly sent from visit 2. Serious adverse events will be followed by active surveillance, on each visit, and by direct contact of the research team if a visit has been missed. Serious AE are defined as any adverse event that resulting in death, hospitalization, permanent damage, requirs treatment in the emergency room or life threatening.
Secondary outcomes: T-cell activity as in detailed methods appendix.
SARS-CoV-2 cumulative incidence and vaccine efficacy will be assessed, by active surveillance of weekly SARS-CoV-2 PCR testing regardless of any symptom. Additionally, symptoms will be assessed on all visits and participants will be encouraged to perform home antigen testing if any symptom occurs in between PCR testing, or upon exposure to SARS-CoV-2 detected individuals.
All data regarding participants would be stored in secured computers and would be available only for this specific study purpose. Any data transferred to a third party will be de-identified, the identification key will be kept by the PI.

Statistical Analysis Plan:
Sample size: To identify a 2-fold difference, in GMT of IgG between the two intervention groups, with alpha of 0.05 and beta of 0.8, 65 participants in each group are needed. To identify a 3-fold difference before and after the fourth dose for each group, 190 participants are needed. To detect a 20% difference in rate of adverse events between the two intervention arms, we will need 108 participants in each group.
To calculate cumulative incidence subjects will be included in follow up starting from day 8 after receiving the fourth dose. For Control participants, the start day will be on the day the matched intervention arm participant entered the study. Follow up will continue until the end of the study or until becoming positive. Analysis will be performed using two methods: Poisson regression with vaccine groups as the main covariates and calendar day and age-group as confounder covariates; and Cox regression, with calendar days for baseline hazard, left truncation for persons in the vaccine groups who are vaccinated later than January 3 rd , including vaccine groups as the main covariates and age-group as confounding covariates. In secondary analyses, the effect of vaccine in two separate periods will be evaluated: from 8-14 days following vaccination and from 15-29 days following vaccination.

Benefits of participating in the study:
The potential benefit of improved protection from SARS-CoV-2. Currently, according the effectiveness of the previous doses, and in the absence of effective drug therapy, it is expected that an additional dose will raise the level of antibodies and thus raise the protection from SARS-CoV-2.

Risks in participating in the study:
The known previous adverse events are expected to occur, these included frequent local reactions (mostly local pain), as well as systemic reactions including fatigue, fever, lymphadenopathy, myalgia, these have typically lasted up to 48 hours. Rarely, myocarditis has occurred (1:6,000-24,000, mostly in young men). It is yet unknown if a 4th dose will have additional adverse reactions.
Some concerns may be raised regarding potential affinity maturation of B cells specific for ancestral variant epitopes encoded by the vaccine, skewing of the T cell response and depletion of memory T cells against other pathogens. The latter point with respect to T cells is not supported by convincing evidence in humans to suggest that this may be the case in the long term nor that it may be detrimental to human health. Many different vaccines are administered at regular intervals in humans and significant detrimental effects to the immune system's response to specific and other pathogens have not been clearly documented.

Introduction
This statistical analysis plan (SAP) provides detailed methodology for summary and statistical analysis of the data collected in studies 8980-21 and 9035-21.

Study Aims:
The major objective of this study is to describe the immune responses following a 4 th dose of either BNT162b2 or mRNA1273, following 3 doses of BNT162b2, when the third dose was given at least 4 months earlier.
Immune responses to be assessed: The second major objective is to assess safety of a 4 th dose. The following solicited local and systemic adverse events will be reported and the proportion of participants reporting each event among the 4 th dose recipients will be calculated with its 95%CL.
1. Local reactions, including pain, redness, swelling or itching in the injection site.
Unsolicited AE will also be collected on each visit until visit 5.
Secondary aim: to assess incidence and characteristics of breakthrough infections in 4 th dose recipients and compare them to infections in the control group, and between recipients of either vaccine.

Study Design:
This is an open-label, non-randomized trial, to assess the immune response of a 4 th dose of either BNT162b2 or mRNA1273. We plan to enroll 150-200 participants to each arm. A control group, which will not recieive a 4 th dose, but has similar baseline characteristics (from the same eligibile sub-cohort) will be selected for comparisons. For each vaccine recipient 2 matched controls will be selected. Several measures of immunogenicity will be measured and reported: Randomization: This a non-randomized, open-label trial. Designation to an intervention arm will be time dependent. Enrollment to the first intervention arm (BNT162b2) will take place during two consecutive days. Once approval of protocol 9035-21 will be given, two days of enrollment to the second arm will take place.
Control group: From the sub cohort of all eligible individuals , an age matched (+/-5 years age difference allowed), will be selected in a 1:2 ratio to each participant in each of the intervention arms. A single control will be allowed to serve as a matched control of both intervention groups. Perfect age and gender will be preferred. Control group will be encouraged through text messages, e-mails and telephone calls to obtain a once weekly RT-PCR test for COVID-19. They will be instructed to report any positive COVID-19 test obtained regardless of whether it was taken as part of the study. Additionally they will receive a computer based questionnaire to assess their compliance with COVID-19 testing.

Study Objectives and Endpoints:
Objective Estimate Endpoint (outcome measure) Primary To assess immune response to a 4 th COVID-19 mRNA vaccine Geometric mean titer (GMT) at each time point 1) Anti SARS-CoV-2 RBD IgG titers in BAU at each time point. GMT of the groups will be calculated with their 95% confidence interval. 2) Pseudoneutralization titers at each time point for each group will similarly be calculated. 3) Microneutralization titers of Delta, Omicron and WT strains before 4 th dose and on each following visit.
Primary Safety Endpoints are as follows: 1) Immediate Reactions -participants will be followed for 30 min after recipient of the vaccine dose. Any immediate reaction will be reported. Primary endpoints that will be reported: The number of immediate reactions in each group, and proportion of immediate reactions. Comparisons between the groups and between age groups will be performed. 2) Local reactions (Pain, redness, swelling, rash or itching) will be collected via an electronic questionnaire from day 5 to day 21. The following measures will be assessed: a. Presence or absence b. Duration (in days) c. Severity (from mild-1, to severe-10) 3) Systemic AE (Lymph node enlargement, Fever>37.5, fatigue, myalgia, headache, facial nerve palsy, paresthesia, late allergic reaction) will be similarly collected via the electronic questionnaire from day 5 to 21. The following measures will be assessed: a. Presence or absence b. Duration(in days) c. Severity (from mild-1, to severe-10), severity will be divided to three categories-1-3-mild, 4-6 moderate, 7-10 severe 4) Serious AE will be collected through the 180 days of the study. Will be categorized according the MedDRA terms. The safety endpoints "SAEs from the vaccination (received in this study) through 6 months after the vaccination" will be summarized by system organ class and preferred term. Additionally, SAEs will be listed.

Secondary endpoints:
Secondary Immunogenicity Endpoints: 1) Anti SARS-CoV-2 IgA at each time point. GMT of the groups will be calculated with their 95% confidence interval. 2) T cell activity, measured as Tcell activity/10 6 cells. Will be assessed on visits 1, 3, 5 and 7.

Secondary SARS-CoV-2 infection Endpoints
3) SARS-CoV-2 PCR will be performed by nasopharyngeal-oropharyngeal swabs in each visit, and while the pandemic surge is high (>10k newly detected cases/d) active weekly surveillance will continue, for both intervention and control arms. 4) Symptomatic COVID -any SARS-CoV-2 symptom will be requested to be reported.
Upon reporting of such a symptom, rapid Ag test and PCR will be performed.

Exploratory endpoints:
Bcell repertoire -initially, on visits 1, 3, 5 and 7, whole blood will be drawn PBMCs separated, and frozen for later evaluation of the B cell repertoire

Baseline variables:
Measurements or samples collected prior to the study vaccination in this study period are considered the baseline data for the assessments.
Demographics, Medical and vaccination History -have been collected upon initial enrollment to the HCW serology study. Yet, the general comorbidity computer-based questionnaire will be filled upon enrollment to this study once again, to reassure full data availablility. These data include date of birth, sex (male or female) , height and weight, comorbidities including hypertention, dyslipidemia, autoimmune disease, diabetes, heart disease, lung disease, coagulation disorder, immunosuppression (including organ transplant recipient, currently undergoing biologic therapy/chemotherapy, treated with corticosteroids, underwent a splenectomy, or diagnosed with HIV), liver disease and kidney disease.
Physical exam by physician / nurse, on vaccination day, including BP, pulse, will be measured following vaccine administration, and if any immediate reaction will develop.

Sample Size and Power
To identify a 2-fold difference, in GMT of IgG between the two intervention groups, with alpha of 0.05 and beta of 0.8, 65 participants in each group are needed. To identify a 3-fold difference before and after the fourth dose for each group, 190 participants are needed. To detect a 20% difference in rate of adverse events between the two intervention arms, we will need 108 participants in each group.

Safety Monitoring Committee reports:
Following each visit a Safety monitoring report will be sent to the committee for evaluation.

Immunogenicity Review
Due to the urgency to inform public health decisions. Following each timepoint, within 2 weeks, as the primary outcomes will be analysed and reported as needed. Data will be disseminated to public health officials as needed and presented to inform the scientific community.

General Analyses
The following descriptive statistics will be used to summarize continuous variables: number of non missing values, mean, standard deviation, median, range.
For binary variables: descriptive statistics for categorical variables (proportions) will be presented in percentage, and the 95%CI, when applicable.
For antibody titers the geometric means will be calculated as the mean of the assay results after making the logarithmic transformation and then exponentiating the mean to express results on the original scale. Two sided 95% CI will be obtained by taking log transformation of assay results, calculating the 95% CI with reference to students t-distribution and then exponentiating the confidence limits.
Geometric mean fold rises -(GMFR) are defined as ratios of the results after vaccination to the results before vaccination. GMFR are limited to participants with nonmissing values at both time points. They are calculated as the mean of the difference of logarithmically transformed assay result and exponentiating the mean. The associated 2-sided 95% Cis will be obtained by constructing Cis using students t-distribution and then exponentiating the confidence limits.
Geometric mean ratios -will be calculated as the mean of the difference of logarithmically transformed assay results and exponentiating the mean. Two-sided Cis will be obtained.

Methods to manage missing data
Participants will receive 4 adverse events electronic questionnaires, participants who will not reply to any of the questionnaires will be contacted by telephone to answer a phone questionnaire, participants who will not reply to any of the questionnaires will be considered as missing data.
Missing serology results will not be imputed.
No additional imputation will be applied to other missing data.
Graphical summaries of the data will be presented using graphpad, including bar plots, scatter plots or line plots.